This proposal focuses on the analysis of the molecular mechanisms involved in determination of the dorsoventral axis in Xenopus embryos. The Wnt family of secreted proteins are known to specify cell fates and embryonic polarity in many developmental processes, including specification of the dorsoventral polarity in vertebrates. Recent studies point to the cytoplasmic proteins Dishevelled and Axin as the key regulators of the Wnt pathway. Our preliminary results indicate that a novel maternal Xenopus homologue of Axin (Xaxin) is directly regulated by Dishevelled. To further investigate the molecular mechanisms of signal transduction through Axin and Dishevelled, we are using the Xenopus system, in which we plan to combine biochemical analysis with functional embryological studies. Physical interactions of Xaxin and Dishevelled will be analyzed in immunoprecipitaion and subcellular colocalization studies. Specific domains of Xdsh and Xaxin, that are involved in complex formation will be identified and their role in Wnt signal transduction will be evaluated by functional analysis of mutated proteins by microinjections. These studies will assess whether Wnt signaling regulates the interaction of Xdsh and Xaxin. In situ hybridization and immunocytochemistry will be used to assess the distribution of Xaxin RNA and protein in the early embryo and to evaluate whether Xaxin is spatially controlled or post-translationally modified during the specification of the dorsoventral axis. While in the embryo the Wnt pathway is essential for the establishment of the dorsoventral axis in vertebrate embryos, several components of this pathway are known to be deregulated during cancer in humans. Thus, studying the mechanisms of Wnt signal transduction, we hope to get insight into basic questions of axis specification and into molecular mechanisms of cancer.